Enteroviruses are among the most severe causes of human disease, and recent outbreaks of emerging enteroviruses such as types D68 and 71 have highlighted the need to understand more about these pathogens. Our comparative studies of poliovirus (PV), coxsackievirus B3, and now EV-D68 have shown many commonalities in the life-cycles of these viruses. We have focused our studies on how members of the Enterovirus genus trigger a cellular pathway known as autophagy to promote production of infectious virus. Autophagy is a constitutive degradative cellular process required for turnover of damaged vesicles, aggregated proteins, and other spent cellular components. During times of stress, including amino acid starvation, organismal development, and infection, autophagy is up-regulated. Autophagic vesicles, thought to be derived from the complex web of viral RNA replication membranes, can be observed during mid-to-late infection. We and others have demonstrated that viral proteins specifically induce autophagic signaling and autophagosome formation. These autophagosomes acidify, promoting maturation of the viral capsid and release of newly formed infectious viruses, often encased in these cell-derived membranes, from the cell. In this proposal, we outline a comprehensive plan to understand generation of membranes during infection, from the initial RNA replication membranes to the autophagosomes promoting maturation of virions to the single-membraned virus- containing vesicles being released from the cell. Understanding this entire pathway, the latter parts of which have only been identified within the past few years, will provide an understanding of how these viruses replicate, mature, release from cells, and evade immune detection. In Specific Aim I, we will analyze how specific viral non-structural proteins initiate autophagosome formation at the early stages of infection and regulate downstream steps in autophagosome formation, acidic maturation, and virus release. In Specific Aim II, we will investigate the viral and host requirements for development of viral RNA replication membranes, and the mechanism of formation of autophagosomes from these convoluted membrane structures, during infection. These experiments can be carried out in the absence of host proteins from the autophagic pathway and other cellular pathways. In addition, we have shown that acidic autophagosomes promote maturation of the virion by inducing cleavage of the capsid protein VP0 into VP2 and VP4. We propose a biochemical approach to identifying the factor(s) contained within autophagosomes required for infectious maturation of viral capsids. There is a major research gap in understanding development of enterovirus-induced membranes, from the initial signals sent by virus proteins at the beginning of infection, to the release of virus-containing vesicles. This proposal will address that need by investigating each step in this pathway from beginning - initiation of RNA replication membranes - to end - the release of virus-containing vesicles.